Broadly neutralizing antibodies (bnAbs) are expected to be a crucial component of vaccine-elicited protective immunity against HIV-1. However, all attempts to elicit such responses to date have failed. The lack of progress in this area could relate to the insufficient authenticity of candidate immunogens. A consistent finding of immunogenicity studies has been that while immune sera efficiently bind to the index immunogen, few Abs recognize the native form of Envelope glycoprotein (Env) found on virus particles. Because nAbs have the select ability to bind to these Env trimers, we hypothesize that Env trimers may in turn be the only antigen capable of selectively and reliably inducing nAbs in a vaccine setting. Testing this hypothesis has until now been impossible, due to problems in isolating pure native Env trimers. For example, particulate vaccines bear many non-functional forms of Env on their surfaces, as well as native trimers. These alternative forms of Env are "promiscuous", in that they are recognized by non-neutralizing Abs (non-nAbs). As a result, they interfere with the development of nAbs. Here we describe new methods to completely eliminate non-functional Env, which will allow us to isolate and test pure authentic Env trimers as vaccines. Our Specific Aims are: Specific Aim 1: To produce virus-like particles bearing authentic Env trimers as the sole form of Env. We are attempting to eliminate non-functional Env from particles, leaving only intact trimers. New strategies have brought us close to achieving this goal. Specific Aim 2: To generate soluble forms of pure authentic Env trimers. We are investigating methods to purify authentic soluble Env trimers. The antigenic topology and stability of pure soluble trimers will be characterized. Specific Aim 3: To evaluate pure authentic trimer immunogens in rabbits. The results from immunizations of successive groups of animals will guide improvements in the consistency, magnitude and breadth of nAb responses. The kinetics, duration and specificity of nAb responses will be characterized. Specific Aim 4: To rescue broadly neutralizing monoclonal antibodies using pure trimer VLPs as "bait". BnmAbs are useful tools for vaccine design. Pure authentic Env trimers may be ideal "baits" for selecting memory B cells, allowing new bnmAbs to be rescued. We will generate fluorochrome-labeled baits and work in collaboration with a group who has recently demonstrated a flow cytometry method to rescue new bnmAbs from HIV-1-infected donors. The neutralization potency, breadth and specificity of any newly identified bnmAbs will be determined. We will also examine IgG sequences and the extent of divergence from the germline. PUBLIC HEALTH RELEVANCE: We hypothesize that pure native HIV-1 Env trimers can elicit effective nAb responses. We will use a new strategy to produce VLPs bearing only authentic Env trimers. By similar methods, we will generate fully authentic soluble Env trimers. Their ability to elicit nAbs will be compared to mock immunogens.